Cell death is an important aspect during the embryonic or post-natal development of major organ systems. Apoptosis, or programmed cell demise, also plays a critical role in maintaining homeostasis in many adult tissues. Within vertebrates, bcl-2 is the best understood gene in a cell death pathway and functions as a cell death repressor.
Apoptosis is a term used to refer to the process(es) of programmed cell death and has been described in several cell types (Waring et al. (1991) Med. Res. Rev. 11: 219; Williams G. T. (1991) Cell 65: 1097; Williams G. T. (1992) Trends Cell Biol. 2: 263; Yonisch-Rouach et al. (1991) Nature 352: 345). Apoptosis is likely involved in controlling the amount of and distribution of certain differentiated cell types, such as lymphocytes and other cells of the hematopoietic lineage. The mechanism(s) by which apoptosis is produced in cells is incompletely understood, as are the regulatory pathways by which the induction of apoptosis occurs.
Apoptosis was first described as a morphologic pattern of cell death characterized by cell shrinkage, membrane blebbing and chromatin condensation culminating in cell fragmentation (Kerr et al., 1992). One hallmark pattern early in the process of cell death is internucleosomal DNA cleavage (Wyllie, 1980). The death-sparing effects of interrupting RNA and protein synthesis and the stereotyped patterns of cell death during development were consistent with a cell autonomous genetic program for cell death (Wyllie et al. (1980) Int. Rev. Cytol. 68: 251; Sulston, J. and Horvitz, H. (1977) Develop. Biol. 56: 110; Abrams et al. (1993) Development 117: 29). The isolation of mutants defective for development cell death in the nematode Caenorhabditis elegans supported this view (Ellis, H. and Horvitz, H. (1986) Cell 44: 817; Hengartner et al. (1992) Nature 365: 494).
The consistency of the morphologic and biochemical patterns defined as apoptosis within different cell types and species, during normal development and as a response to external stimuli are consistent with a common cause of cellular mortality. This thesis is supported by the concept of an endogenous program responsible for cell death and the presence of gene products which are positive and negative regulators of apoptosis. The best studied negative regulator of apoptosis is the bcl-2 proto-oncogene product. It provides the strongest evidence for a shared mammalian pathway of death by its ability to block a wide variety of cell death models.
This pattern of morphologic cell death is characterized by a dramatic plasma membrane blebbing, cell volume contraction, nuclear pyknosis, and internucleosomal DNA degradation following the activation of an endonuclease. Over expression of mitochondrial bcl-2 appears to function as an antidote to this process and has the unique function of blocking programmed cell death independent of promoting proliferation.
The maintenance of homeostasis in normal tissue, in many respects, reflects a simple balanced equation of input (cellular proliferation and renewal) versus output (cell death). This is most easily envisioned for encapsulated organs, such as the prostate, but is also true of the recirculating hematopoietic lineages. The maintenance of remarkably invariant cell numbers reflects tightly regulated death pathways as well as controlled proliferation. See for example S. J. Korsmeyer “bcl-2 Initiates a New Category of Oncogenes: Regulators of Cell Death”, Blood Vol. 80 No. 4 pp. 879-886, Aug. 15, 1992.
Programmed cell death represents a cell autonomous suicide pathway that helps restrict cell numbers. The well-defined loss of specific cells is crucial during embryonic development as part of organogenesis. In the mature tissues, genetically programmed demise regulates the volume of cells. A morphologically distinct and temporally regulated cell death entitled apoptosis has been identified by Wyllie A. H.: “Apoptosis: Cell death in tissue regulation”. J. Pathol 153: 313, 1987. Cells dying by apoptosis display marked plasma membrane blebbing, volume contraction, nuclear condensation, and the activation of an endonuclease that cleaves DNA into nucleosomal length fragments.
The genetic regulation of cell death is thought to be a central mechanism of cellular homeostasis and development (Bernardi, P. et al, 1999, Bossy-Wetzel, E. et al, 1998, Boyd, J. M. et al, 1994, Chautan, M. et al, 1999). The Bcl-2 family of genes (Bernardi, P. et al, 1999, Chen, G. et al, 1999), which are related to ced-9 of C. elegans (Chen, G. et al, 1997), were originally identified as repressors of cell death. It is known that both pro-and anti-apoptotic Bcl-2 homologs exist, however their exact biochemical function has not been determined. Recent studies suggest that Ced-9 and Bcl-2/BCl-XL may physically interact with proteins that are required for the execution of apoptosis, Ced-3 and Ced-4 (Chi, S. et al, 1999, Crompton, M. 1999, Datta, S. R. et al, 1997), however these proteins have not been isolated and purified. Ced-3 is a protease which in mammals is represented by a large family of cysteine proteases which cleave after aspartic acid, now called caspases (Chautan, M. et al, 1999, Deas, O. et al, 1998). In mammalian cells overexpression of bcl-2 prevents the processing and activation of caspase-3 (CPP32) (Earnshaw, W. C. et al, 1999, Finucane, D. M. et al, 1999).
Bcl-2 family members bear C-terminal transmembrane domains that allow their association with the outer mitochondrial membrane (Goping, 1. S. et al, 1998) and this mitochondrial localization is important for the suppressive function of Bcl-2 (Green, D. R. et al, 1998, Griffiths, G. J. et al, 1999). There is growing evidence that mitochondrial function is disturbed early in the apoptotic response and may be important in mediating apoptosis (Gross, A. et al, 1999, Hakem, R. et al, 1998, Harada, H. et al, 1999). This is often seen as the loss of mitochondrial membrane potential (Gross, A. et al, 1999, Hakem, R. et al, 1998) and the release of cytochrome c (Harada, H. et al, 1999), and cytochrome c has been implicated in the activation of caspase (Harada, H. et al, 1999, Horvitz, H. R. 1999, Imazu, T. et al, 1999). Bcl-2 can suppress the release of cytochrome c from mitochondria and prevent caspase activation (Horvitz, H. R. 1999, Imazu, T. et al, 1999).
Additionally, the protein encoded by the bcl-2 proto-oncogene has been reported to be capable of inhibiting apoptosis in many hematopoietic cell systems. The proto-oncogene bcl-2 was isolated and characterized as a result of its frequent translocation adjacent to the immunoglobulin heavy chain enhancer in the t (Green, D. R. et al, 1998; Harada, H. et al, 1999) chromosome translocation present in more than 80% of human follicular lymphomas (Chen-Levy et al. (1989) Mol. Cell. Biol. 9: 701; Clearly et al. (1986) Cell 47: 19). These neoplasias are characterized by an accumulation of mature resting B cells presumed to result from a block of apoptosis which would normally cause turnover of these cells. Transgenic mice expressing bcl-2 under the control of the Eu. enhancer similarly develop follicular lymphomas which have a high incidence of developing into malignant lymphomas (Hockenbery et al. (1990) Nature 348: 334; McDonnell T. J. and Korsmeyer S. J. (1991) Nature 349: 254; Strasser et al. (1991) Cell 67: 889).
The capacity of bcl-2 to enhance cell survival is related to its ability to inhibit apoptosis initiated by several factors, such as cytokine deprivation, radiation exposure, glucocorticoid treatment, and administration of anti-CD-3 antibody (Nunez et al. (1990) op. cit; Hockenbery et al. (1990) op. cit; Vaux et al. (1988) op. cit; Alnemri et al. (1992) Cancer Res. 52: 491; Sentman et al. (1991) Cell 67: 879; Strasser et al. (1991) op. cit). Upregulation of bcl-2 expression also inhibits apoptosis of EBV infected B-cell lines (Henderson et al. (1991) Cell 65: 1107). The expression of bcl-2 has also been shown to block apoptosis resulting from expression of the positive cell growth regulatory proto-oncogene, c-myc, in the absence of serum or growth factors (Wagner et al. (1993) Mol. Cell. Biol. 13: 2432). However, the precise mechanism (s) by which bci-2 is able to inhibit apoptosis is not yet fully defined.
The bcl-2 proto-oncogene is rather unique among cellular genes in its ability to block apoptotic deaths in multiple contexts (Korsmeyer, S. (1992) Blood 80: 879). Overexpression of bcl-2 in transgenic models leads to accumulation of cells due to evasion of normal cell death mechanisms (McDonnell et al. (1989) Cell 57: 79). Induction of apoptosis by diverse stimuli, such as radiation, hyperthermia, growth factor withdrawal, glucocorticoids and multiple classes of chemotherapeutic agents is inhibited by bcl-2 in vitro models (Vaux et al. (1988) Nature 335: 440; Tsujimoto, Y. (1989) Oncogene 4: 1331; Nunez et al. (1990) J. Immunol. 144: 3602; Hockenbery et al. (1990) Nature 348: 334; Sentman et al. (1991) Cell 67: 879; Walton et al. (1993) Cancer Res. 53: 1853; Miyashita, T. and Reed, J. (1993) Blood 81: 151). These effects are proportional to the level of bcl-2 expression. Additionally, the endogenous pattern of bc1-2 expression is highly suggestive of a role in the regulation of cell survival in vivo (Hockenbery et al. (1991) Proc. Natl. Acad. Sci. USA 88: 6961; LeBrun et al. (1993) Am. J. Pathol. 142: 743). The bcl-2 protein seems likely to function as an antagonist of a central mechanism operative in cell death.
Additionally, Kerr et al. (Kerr, J. F. R. et al, 1972), on the basis of distinct morphological criteria, identified apoptosis as a programmed and intrinsic cell death pathway, in contrast to necrosis, which was viewed as a passive response to injury. It is now clear that apoptosis is a highly regulated genetic program that is evolutionarily conserved in multicellular organisms and is essential for development and tissue homeostasis (Horvitz, H. R. 1999, 57). The genetic program results in the activation of cysteine aspartyl proteases (caspases) that cleave nuclear and cytoplasmic substrates and disassemble the cell (Earnshaw, W. C. et al, 1999, 54), yielding the characteristic morphological features such as chromatin condensation, DNA fragmentation, plasma membrane blebbing, and the formation of apoptotic bodies (Xue, L. Z., et al., 1999). In contrast to apoptosis, necrosis is considered an unregulated process occurring in response to toxicants and physical injury. This form of cell death is morphologically characterized by extensive mitochondrial swelling, cytoplasmic vacuolation, and early plasma membrane permeability without major nuclear damage (Kerr, J. F. R. et al, 1972, Kitanaka, C. et al, 1999, 55).
Mitochondria appear to play a central role in the induction of cell death. This is thought to occur by at least three possible mechanisms: (i) release of apoptogenic proteins that facilitate caspase activation, (ii) disruption of electron transport, oxidative phosphorylation, and ATP production that can result in an energetic catastrophe, and (iii) alteration of the redox potential, resulting in increased cellular oxidative stress (Green, D. R. et al, 1998). The main biochemical determinant of apoptosis is the activation of caspases, and this is in part regulated by mitochondria. All caspases are synthesized as an inactive polypeptide (zymogen) that must be proteolytically processed to form an active tetramer (Earnshaw, W. C. et al, 1999). Recent work proposes that this processing is initiated through autocatalytic activation. For example, the caspase 8 zymogen is aggregated for autoprocessing by ligand-induced clustering of trimeric death receptors such as CD95/Fas (Srinivasula, S. M., et al., 1998). Active caspase 8 cleaves the proapoptotic BCL-2 family member BID, which is then able to translocate to mitochondria (Li, H. et al, 1998, Luo, X. et al, 1998). BID, as well as many other apoptotic signals, induces mitochondria to release cytochrome c, which functions as a cofactor with dATP for Apaf-1 binding and activation of caspase 9 and downstream effector caspases (Li, P. et al, 1997, 51). Another less well studied mitochondrial apoptogenic protein is apoptosis-inducing factor (AIF), a flavoprotein released in response to apoptotic signals that translocates to the nucleus to induce DNA fragmentation and chromatin condensation in a caspase-independent manner (Tsujimoto, Y. 1997).
Apoptotic cell death signals induce other mitochondrial changes, such as opening of the permeability transition (PT) pore, a putative highly regulated ion channel located at the contact sites between the inner and outer mitochondrial membrane (Crompton, M. 1999). The PT pore is a large protein complex, primarily composed of the adenine nucleotide transporter (ANT), cyclophilin D, and voltage dependent anion channel (VDAC [also called porin]), that can interact with several other proteins (Crompton, M. 1999, Kroemer, G. et al, 1998). When the PT pore is in the open state, it permits the passage of solutes of; 1,500 Da and results in depolarization of mitochondria, which consequently decreases the measured proton electrochemical gradient (Dcm). This, in turn, can lead to the inhibition of respiration, generation of reactive oxygen species (ROS), and loss of ATP production (Bernardi, P. et al, 1999, Crompton, M. 1999). PT pore opening also increases the permeability of certain ions across the mitochondrial membrane, resulting in increased water influx into the matrix and consequent large-amplitude mitochondrial swelling (Gross, A. et al, 1999, Lemasters, J. J. et al, 1998).
The biochemical determinants of necrotic cell death are less well defined, but similar to apoptosis. It has been suggested that the PT pore might play a major role in necrosis. PT pore opening has been described in response to a rise in cytosolic free Ca 21, anoxia, and reperfusion oxidative stress with overproduction of ROS in cardiac myocytes (Crompton, M. 1999). Although both apoptosis and necrosis are associated with PT pore opening, necrosis is distinguished by an early loss of plasma membrane integrity and ATP, whereas both are maintained and ATP production is required for apoptosis (Leist, M. et al, 1997, Nicotera, P. et al, 1998).
In 1996 Dr. A. H. Greenberg's lab, isolated a protein called BNIP3 and soon thereafter determined that a homodimeric complex of BNIP3 was associated with the energy producing organelle of the cell, the mitochondria, and that BNIP3 had a function in cell death (Chen G et al, 1997). Along with human BNIP3, other family members NIX and NOX (NINA) were identified, as were homologues from other species namely mouse and c. elegans (Chen G et al, 1999; Cizeau J et al, 2000). Initially it was believed that BNIP3 in apoptosis; however, subsequent work on the biological mechanism of BNIP3 revealed that this was inaccurate (Vande Velde C et al, 2000).
It would therefore be useful to determine the role BNIP3 plays in cell death. It would also be useful to develop methods of using BNIP3 to treat diseases.